The present invention relates to combination drug therapy for the treatment of Gaucher""s disease and other glycolipid storage diseases.
Gaucher""s disease is a glycolytic storage disease caused by a genetic deficiency in activity of the catabolic enzyme beta-glucocerebrosidase. Beutler, Proc. Natl. Acad. Sci. USA. 90, 5384-5390 (1993). Manifestations of this disease are impaired hematopoiesis, bone fractures, a thinning of the bone cortex and massive enlargement of the spleen and liver.
In recent years, several therapies have been proposed for the treatment of Gaucher""s disease. An early therapeutic approach involved replacement of the deficient enzyme. See, for example, Dale and Beutler, Proc. Natl. Acad. Sci. USA 73, 4672-4674 (1976); Beutler et al., Blood 78, 1183-1189 (1991); and Beutler, Science 256, 794-799 (1992).
Leading commercial products for enzyme replacement are CEREDASE (glucocerebrosidase), which is derived from human placental tissues, and CEREZYME (recombinant human glucocerebrosidase), both of which are produced by Genzyme Corp. See, for example, U.S. Pat. Nos. 3,910,822; 5,236,838; and 5,549,892. See also U.S. Pat. Nos. 5,879,680 and 6,074,684 on cloned DNA for synthesizing human glucocerebrosidase.
Conjugates of the glucocerebrosidase enzyme with polyethylene glycol (PEG) have also been advanced by Enzon Inc. for treatment of Gaucher""s disease. See, for example, U.S. Pat. Nos. 5,705,153 and 5,620,884.
Still another approach for treatment of the disease is gene therapy, which involves an ex vivo gene transfer protocol. See, for example, U.S. Pat. No. 5,911,983.
Another recent approach involves administration of the totally synthetic drugs, N-butyldeoxynojirimycin and N-butyldeoxygalactonojirimycin, as described, respectively, by Platt et al., J. Biol. Chem. 269, 8362-8365 (1994); Id. 269, 27108-27114 (1994). See also, U.S. Pat. Nos. 5,472,969; 5,786,368; 5,798,366; and 5,801,185.
N-butyldeoxynojirimycin (N-butyl-DNJ) and related N-alkyl derivatives of DNJ are known inhibitors of the N-linked oligosaccharide processing enzymes, xcex1-glucosidase I and II. Saunier et al., J. Biol. Chem. 257, 14155-14161 (1982); Elbein, Ann. Rev. Biochem. 56, 497-534 (1987). As glucose analogs, they also have potential to inhibit glycosyltransferases. Newbrun et al., Arch. Oral Biol. 28, 516-536 (1983); Wang et al., Tetrahedron Lett. 34, 403-406 (1993). Their inhibitory activity against the glycosidases has led to the development of these compounds as antihyperglycemic agents and as antiviral agents. See, e.g., PCT Int""l. Appln. WO 87/030903 and U.S. Pat. Nos. 4,065,562; 4,182,767; 4,533,668; 4,639,436; 5,011,829; 5,030,638; and 5,264,356.
In particular, N-butyl-DNJ has been developed as an inhibitor of human immunodeficiency virus (HIV) as described by Fleet et al., FEBS Lett. 237, 128-132 (1988), and by Karpas et al., Proc. Nat""l. Acad. Sci. USA 85, 9229-9233 (1988), U.S. Pat. No. 4,849,430; and as an inhibitor of hepatitis B virus (HBV) as described by Block et al., Proc. Natl. Acad. Sci. USA 91, 2235-2239 (1994), PCT Int""l. Appln. WO 95/19172 and U.S. Pat. No. 6,037,351.
In accordance with the present invention, a novel method and composition is provided for the treatment of a patient affected with Gaucher""s disease or other such glycolipid storage diseases. The method of the invention comprises administering to said patient a therapeutically effective amount of both a N-alkyl derivative of 1,5-dideoxy-1,5-imino-D-glucitol having from about two to about 20 carbon atoms in the alkyl chain and a glucocerebrosidase enzyme. The N-alkyl substituent can be a short-chain alkyl group such as, e.g., ethyl, butyl or hexyl, or a long-chain alkyl group such as, e.g, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, octadecyl and eicosyl.
A therapeutically effective amount is meant an amount effective in alleviating or inhibiting Gaucher""s disease or other such glycolipid storage diseases in said patient. The glucocerebrosidase provides enzyme replacement for non-breakdown of glucocerebroside and the N-alkyl-DNJ jointly provides glycolipid inhibitory action. By use of the combination drug therapy of the invention, the medical benefits of both types of drugs should accrue to the patient with reduced amounts of either or both drugs than otherwise necessary to obtain equivalent or enhanced therapeutic results. That is, an additive or synergistic effect can reduce the frequency of the administration of the glucocerebrosidase enzyme and lower the dose of the long-chain N-alkyl-DNJ otherwise required for monotherapy of the disease.
The alkyl group in the short-chain N-alkyl-DNJ compounds preferably contains four to six carbon atoms (e.g., butyl or hexyl). A most preferred compound is N-butyl-1,5-dideoxy-1,5-imino-D-glucitol, also known as the N-butyl derivative of deoxynojirimycin (DNJ), which also is abbreviated herein as N-butyl-DNJ.
The alkyl group in the long-chain N-alkyl-DNJ compounds preferably contains nine to ten carbon atoms (i.e., nonyl and decyl). A most preferred compound is N-nonyl-1,5-dideoxy-1,5-imino-D-glucitol, also known as the N-nonyl derivative of deoxynojirimycin (DNJ), which also is abbreviated herein as N-nonyl-DNJ.
In the field of general organic chemistry, the long-chain alkyl groups are known to provide more hydrophobic properties to compounds than are the short-chain alkyl groups. That is, solubility with water decreases with increase in chain length and decrease in temperature. For example, at 46xc2x0 C., caproic acid (short-chain hexyl group) dissolves 10% by weight of water, whereas stearic acid (long-chain octadecyl group) dissolves only 0.92% even at the higher temperature of 69xc2x0 C. Bailey""s Industrial Oil and Fat Products, ed. Daniel Swern, 3d ed. 1964, p. 126.
The long-chain N-alkyl derivatives of DNJ are known amino-sugar compounds. They were originally described as members of a group of short-chain and long-chain N-alkyl derivatives of DNJ having both glucosidase I inhibitory activity and antiviral activity, although no data on the long-chain N-alkyl derivatives was disclosed. See, e.g., DE 3,737,523, EP 315,017 and U.S. Pat. Nos. 4,260,622; 4,639,436; and 5,051,407.
In another early study, although N-alkylation of the base DNJ reduced the concentration required for 50% inhibition of glucosidase I, the inhibitory activity was reduced as the length of the N-alkyl chain was increased from N-methyl to N-decyl according to Schweden et al., Arch. Biochem. Biophys. 248, 335-340, at 338 (1986).
As far as the antiviral activity of the amino-sugar compounds against any particular virus is concerned, the activity of any specific analog cannot be predicted in advance. For example, in biologic tests for inhibitory activity against the human immunodeficiency virus (HIV), slight changes in the structure of the N-substituent were shown to have pronounced effects upon the antiviral profile as reported by Fleet et al., FEBS Lett. 237, 128-132 (1988). As disclosed in U.S. Pat. No. 4,849,430, the N-butyl derivative of DNJ was unexpectedly found to be more than two log orders more effective as an inhibitor of HIV than the N-methyl analog and three log orders more effective than the N-ethyl analog.
In another study of N-alkyl derivatives of DNJ for activity against glycolipid biosynthesis, the N-hexyl derivative of DNJ required a dose of 0.2 mg/ml, whereas the corresponding N-butyl analog required a dose of only 0.01-0.1. On the other hand, the N-methyl analog was inactive. Thus, it was believed that effective carbon chain length of the N-alkyl group for this activity ranged from 2 to 8 according to U.S. Pat. No. 5,472,969. No disclosure was made therein concerning the N-nonyl or other long-chain N-alkyl derivatives of DNJ.
N-nonyl-DNJ has been reported to be effective as an inhibitor of the Hepatitis B virus (HBV) based on inhibition of alpha-glucosidases in the cellular endoplasmic reticulum (ER) according to Block et al., Nature Medicine 4(5) 610-614 (1998).
The effectiveness of the long-chain N-alkyl derivatives of DNJ in the method of the invention for treatment of Gaucher""s disease and other such glycolipid storage diseases is illustratively demonstrated herein by inhibitory activity of N-nonyl and N-decyl DNJs against glycolipid biosynthesis in Chinese hamster ovary (CHO) cells and human myeloid (HL-60) cells.
CHO cells are well-known glycoprotein-secreting mammalian cells. A typical CHO cell line is CHO-K1 which is available to the public from the American Type Culture Collection, Bethesda, Md., under accession number ATCC CCL 61.
HL-60 cells are human promyelocytic cells described by Collins et al., Nature 270, 347-349 (1977). They are also readily available from the American Type Culture Collection under accession number ATCC CCL 240.
Effective activity of N-nonyl-DNJ also is further illustratively demonstrated herein in conventional bovine kidney cells (e.g., MDBK, ATCC CCL 22) and hepatoma cells (e.g., HepG2, ATCC HB 8065).
The unpredictability of the N-nonyl-DNJ against glycolipid biosynthesis is demonstrated herein by its inhibitory activity in the foregoing two cell lines. The N-nonyl-DNJ was unexpectedly found to be from about ten- to about twenty-fold better in the CHO cells and about four hundred times better in the HL-60 cells than N-butyl-DNJ at equivalent concentrations. The N-decyl-DNJ was demonstrated to be an effective inhibitor in HL-60 cells at 50 times lower concentrations than N-butyl-DNJ. These results were further unexpected in view of the increased hydrophobic nature of the long-chain N-alkyl derivatives of DNJ.
The N-nonyl-DNJ also exhibits a more dramatic difference than N-butyl-DNJ in uptake which permits its use at a substantially lower level. In tests of organ distribution, the N-nonyl-DNJ was taken up five times better into the brain than N-butyl-DNJ. Thus, the N-nonyl-DNJ is believed to be a substantially better compound than N-butyl-DNJ for treating glycolipid storage disorders which involve the non-systemic side.
N-nonyl-DNJ and N-decyl-DNJ can be conveniently prepared by the N-nonylation or N-decylation, respectively, of 1,5-dideoxy-1,5-imino-D-glucitol (DNJ) by methods analogous to the N-butylation of DNJ as described in Example 2 of U.S. Pat. No. 4,639,436 by substituting an equivalent amount of n-nonylaldehyde or n-decylaldehyde for n-butylaldehyde. The starting materials are readily available from many commercial sources. For example, DNJ is available from Sigma, St. Louis, Mo., whereas n-nonylaldehyde, also known as 1-nonanal or pelargonaldehyde, and n-decylaldehyde, also known as decanal, are commercially available from Aldrich, Milwaukee, Wis. It will be appreciated, however, that the N-alkyl-DNJ used in this combination drug therapy is not limited to any particular method of synthesis of the N-butyl-DNJ, N-nonyl-DNJ, N-decyl-DNJ, or other N-alkyl derivatives of DNJ.
The glucocerebrosidase used in the combination drug therapy also is a known drug as described above. For example, it can be derived from human placental tissue by conventional isolation and purification techniques or prepared by recombinant DNA procedures. Conventional methods of isolation and purification from human placental tissue are described By Dale and Beutler, Proc. Natl. Acad. Sci. USA 73, 4672-4674 (1976) and in U.S. Pat. No. 3,910,822. Suitable methods of production by recombinant DNA are described in U.S. Pat. Nos. 5,236,838, 5,549,892 and 5,879,680. The glucocerebrosidase can also be conjugated with carrier molecules such as, for example, polyethylene glycol (PEG) as described in U.S. Pat. Nos 5,705,153 and 5,620,884. It will be appreciated, however, that the glucocerebrosidase used in the combination drug therapy is not limited to any particular method of production.
The N-butyl-DNJ, N-nonyl-DNJ, N-decyl-DNJ, and other N-alkyl derivatives of DNJ, can be used for treatment of patients afflicted with Gaucher""s disease and other glycolipid storage diseases by conventional methods of administering therapeutic drugs. Thus, the active compound is preferably formulated with pharmaceutically acceptable diluents and carriers. The active drug can be used in the free amine form or the salt form. Pharmaceutically acceptable salt forms are illustrated, e.g., by the HCl salt. The amount of the active drug to be administered must be an effective amount, that is, an amount which is medically beneficial against Gaucher""s disease or other glycolipid storage disease but does not present adverse toxic effects which overweigh the advantages that accompany its use.
It would be expected that the adult human daily dosage would normally range from about 0.1 to about 1000 milligrams of the active compound. The preferable route of administration is orally in the form of capsules, tablets, syrups, elixirs, gels and the like, although parenteral administration also can be used.
The glucocerebrosidase enzyme likewise can be administered by conventional means, preferably by intravenous infusion, e.g. administration of the active enzyme in a pharmaceutically acceptable carrier such as physiological saline. Initially, a dose of about 60 U per kilogram of body weight every two weeks was recommended. See, e.g., Beutler, Science 256, 794-799 (1992). After 6 to 12 months of therapy, doses of 7.5 to 15 U per kilogram every two weeks were suggested according to Moscicki et al., New Engl. J. Med. 328, 1564 (1993).
Illustratively, the two combination drug components can be administered together or separately, e.g., administration of the enzyme by periodic administration (e.g., weekly or bimonthly) and oral administration of the N-alkyl-DNJ daily.
By use of the combination drug therapy described herein, an additive or synergistic effect can be obtained to reduce the aforesaid frequency of the intravenous injection of the glucocerebrosidase and lower the dose of the N-alkyl-DNJ otherwise required for monotherapy of Gaucher""s disease.
Suitable formulations of the active components in pharmaceutically acceptable diluents and carriers in therapeutic dosage from can be prepared by the person skilled in the art by reference to general texts and treatises in the pharmaceutical field such as, for example, Remington""s Pharmaceutical Sciences, Ed. Arthur Osol, 16 ed., 1980, Mack Publishing Co. Easton, PA., and 18th ed., 1990.
Other glycolipid storage diseases to which the method of the invention is directed are, e.g., Tay-Sachs disease, Sandhoff disease, Fabry disease, GM1 gangliosidosis and fucosidosis.